Isolation Of Specific Promoter In Peanut (Arachis Hypogaea L.) And Construction Of Vectors Resistance To Aflatoxin

Peanut (Arachis hypogaea L.) is one of the world’s most important commercial crops, cultivated in over 100 tropical and subtropical countries. Peanut products are widely consumed due to their excellent flavor and nutritional content. However, aflatoxin contamination of peanut has been the worldwide problem that seriously impacts peanut production, trade and food safety. How to solve the aflatoxin contamination problem of peanut is an urgent matter.With the successful application of transgenic technology in many other crops, the transgenic technology has become the most promising method to resolve the aflatoxin contamination problem in peanut. However, as consumer very concerns about the security of transgenic plants, the application of transgenic technology has been slowed in crops such as vegetables, fruits and grain crops. There are three main problems that hinder transgenic development and application in crops:the environmental risks of marker genes; the constitutive expression of exogenous genes in host plant; the potential risk of foreign genes to human.To avoid the above problems of transgenic peanut and to obtain the safe Aspergillus flavus resistance transgenic peanut, the following studies has been done in this study:1. A 1690bp and a 1103bp upstream fragments of an A. flavus induced gene AhOMTl were isolated from peanut by Flanking PCR based on an available fragment cloned before. The AhOMTlgene has no expression in kinds of peanut tissues but showed high expression level in peanut pericarp after A. flavus infection. Bioinformatics analysis showed that there are many cis-elements in the promoter, including fungal elicitor responsive elements, MYB binding site, defense and stress responsiveness elements, phytohormone responsive elements, TATA-box and CAAT-box. This suggested that AhOMT1 promoter may be a fungal-inducible promoter.2. A pericarp-abundant expression promoter was cloned from the upstream of AhLEC1 gene which abundantly express in peanut pericarp and has no expression in kernel, and a pericarp-specific expression promoter was cloned from the upstream of pericarp-specific expressed gene AhLACl of peanut. The two promoters were used to construct tissue-specific expression vectors driving the expression of the resistance related genes of CHI, GLU, TTG2, WRKY, CBF2, RIP, RESS respectively on pBI121. Thirteen plant expression vectors were constructed in this study.3. One of the constructed vector pBI121A-B1-RIP was used to Agrobacterium-mediated genetic transformation of peanut. To improve the survival rate of transgenic peanut plants grafting technique was used to replace the transplant procedure of rooting plants in the study. The putative transgenic plants were identified by PCR technology and three positive transgenic plants were obtained.